The use of genetic recombination technology makes it possible to impart new traits to plants by expressing a useful gene in a target plant. Genetically modified plants produced in this manner have already been cultivated widely. Since regulation of gene expression is mainly controlled at the level of transcription, transcriptional regulation is the most important in terms of regulating the expression of genes. Namely, transcribing a gene at a suitable time, in a suitable tissue and at a suitable strength is important for producing an industrially useful genetically modified plant. In many cases, transcription is controlled by a DNA sequence on the 5′-side of a translated region. A region of DNA that determines the starting site of gene transcription and directly regulates the frequency thereof is referred to as a promoter. A promoter is located several tens of base pairs (bp) from the 5′-side of an initiation codon, and frequently contains a TATA box and the like. A cis element that binds various transcriptional regulatory factors is also present on the 5′-side, and the presence thereof serves to control the timing of transcription, the tissue in which transcription takes place and transcriptional strength. Transcriptional regulatory factors are classified into many families according to their amino acid sequence. For example, examples of well-known families of transcriptional regulatory factors include Myb transcriptional regulatory factors and bHLH (basic helix loop helix) regulatory factors. In actuality, the terms transcriptional regulatory factor and promoter are frequently used with the same meaning.
Anthocyanins, which compose the main components of flower color, are a class of secondary metabolites generically referred to as flavonoids. The color of anthocyanins is dependent on their structure. Namely, color becomes bluer as the number of hydroxyl groups on the B ring of anthocyanidins, which is the chromophore of anthocyanins, increases. In addition, as the number of aromatic acyl groups (such as coumaroyl group or caffeolyl group) that modify the anthocyanin increases (namely, the wavelength of maximum absorbance shifts to a longer wavelength), the color of the anthocyanin becomes bluer and the stability of the anthocyanin is known to increase (see Non-Patent Document 1).
Considerable research has been conducted on those enzymes and genes that encode those enzymes involved in the biosynthesis of anthocyanins (see, Non-Patent Document 1). For example, an enzyme gene that catalyzes a reaction by which an aromatic acyl group is transferred to anthocyanin is obtained from Japanese gentian, lavender and petunias (see Patent Document 1 and Patent Document 2). An enzyme gene involved in the synthesis of anthocyanin that accumulates in the leaves of perilla (malonylshisonin, 3-O-(6-0-(E)-p-coumaroyl-β-D-glucopyranosyl)-5-O-(6-O-malonyl-β-D-glucopyranosyl)-cyanidin) (see Non-Patent Document 2) has previously been reported in hydroxycinnamoyl CoA:anthocyanin 3-glucoside-aromatic acyl transferase (3AT) gene (or more simply referred to as “perilla anthocyanin 3-acyl transferase (3AT) gene”) (see Patent Document 1). Moreover, knowledge has also been obtained regarding the transcriptional regulation (control) of biosynthetic genes of anthocyanins. Cis element sequences bound by Myb transcriptional regulatory factor and bHLH transcriptional regulatory factor are present in the transcriptional regulatory region located on the 5′-side of the initiation codons of these genes. Myb transcriptional regulatory factors and bHLH transcriptional regulatory factors are known to control synthesis of anthocyanins in petunias, maize and perilla (see Non-Patent Document 1).
Promoters (to also be referred to as transcriptional regulatory regions) responsible for gene transcription in plants consist of so-called constitutive promoters, which function in any tissue and at any time such as in the developmental stage, organ/tissue-specific promoters, which only function in specific organs and tissues, and time-specific promoters, which only express genes at a specific time in the developmental stage. Constitutive promoters are frequently used as promoters for expressing useful genes in genetically modified plants. Typical examples of constitutive promoters include cauliflower mosaic virus 35S promoter (to also be abbreviated as CaMV35S) and promoters constructed on the basis thereof (see Non-Patent Document 3), and Mac1 promoter (see Non-Patent Document 4). In plants, however, many genes are only expressed in specific tissues or organs or are only expressed at specific times. This suggests that tissue/organ-specific or time-specific expression of genes is necessary for plants. There are examples of genetic recombination of plants that utilize such tissue/organ-specific or time-specific transcriptional regulatory regions. For example, there are examples of protein being accumulated in seeds by using a seed-specific transcriptional regulatory region.
However, although plants produce flowers of various colors, there are few species capable of producing flowers of all colors due to genetic restrictions on that species. For example, there are no varieties of rose or carnation in nature that are capable of producing blue or purple flowers. This is because roses and carnations lack the F3′5′H gene required to synthesize the anthocyanindin, delphinidin, which is synthesized by many species that produce blue and purple flowers. These species can be made to produce blue flowers by transforming with the flavonoid 3′,5′-hydroxylase gene of petunia or pansy, for example, which are species capable of producing blue and purple flowers. In the case of carnations, the transcriptional regulatory region of chalcone synthase gene derived from snapdragon or petunia is used to transcribe F3′5′H gene derived from a different species. Examples of plasmids containing the transcriptional regulatory region of chalcone synthase gene derived from snapdragon or petunia include plasmids pCGP485 and pCGP653 described in Patent Document 3, and examples of plasmids containing a constitutive transcriptional regulatory region include plasmid PCGP628 (containing a Mac1 promoter) and plasmid pSPB130 (containing a CaMV35S promoter to which is added EI2 enhancer) described in Patent Document 4.
However, it is difficult to predict how strongly such promoters function in recombinant plants to be able to generate a target phenotype. In addition, since repeatedly using the same promoter to express a plurality of foreign genes may cause gene silencing, it is thought that this should be avoided (see Non-Patent Document 5).
Thus, although several promoters have been used to alter flower color, a useful promoter corresponding to the target host plant and objective is still needed.
In particular, chrysanthemum plants (to also be simply referred to as chrysanthemums) account for about 30% of all wholesale flower sales throughout Japan (Summary of 2007 Flowering Plant Wholesale Market Survey Results, Ministry of Agriculture, Forestry and Fisheries), making these plants an important product when compared with roses accounting for roughly 9% and carnations accounting for roughly 7%. Although chrysanthemums come in flower colors including white, yellow, orange, red, pink and purplish red, there are no chrysanthemums that produce bluish flowers such as those having a purple or blue color. Thus, one objective of the breeding of bluish flowers is to stimulate new demand. Chrysanthemum flower color is expressed due to a combination of anthocyanins and carotenoids. Anthocyanins are able to express various colors due to differences in the structure of the anthocyanidin serving as the basic backbone, and differences in modification by sugars and organic acids. However, there are known to be two types of anthocyanins that govern chrysanthemum flower color in which cyanidin at position 3 is modified by glucose and malonic acid (cyanidin 3-O-(6″-O-monomalonyl-β-glucopyranoside and 3-O-(3″,6″-O-dimalonyl-β-glucopyranoside) (see Non-Patent Document 6). In addition, these structures are comparatively simple (see FIG. 1). This causes the range of flower color attributable to anthocyanins in chrysanthemums to be extremely narrow.
As was previously described, although chrysanthemums are the most important flowering plant in Japan, since they are hexaploidal resulting in high ploidy and have a large genome size, in addition to having low transformation efficiency, since they are subject to the occurrence of silencing (deactivation) of transgenes, it is not easy to obtain genetically modified chrysanthemums capable of stable transgene expression. In chrysanthemums transfected with β-glucuronidase (GUS) gene coupled to CaMV35S promoter, the activity of the GUS gene is roughly one-tenth that of tobacco transformed with the same gene, and that activity has been reported to decrease in nearly all individuals after 12 months have elapsed following transformation (see Non-Patent Document 7). Although a promoter of gene that encodes a chlorophyll a/b-binding protein that favorably functions in chrysanthemums has been reported to have been obtained in order to stably express an exogenous gene in chrysanthemums, this promoter is not suitable for expressing genes in flower petals in which there is little chlorophyll present (see Non-Patent Document 8). In addition, when GUS gene coupled to tobacco elongation factor 1 (EF1α) promoter is introduced into chrysanthemums, GUS gene has been reported to be expressed in leaves and petals even after the passage of 20 months or more (see Non-Patent Document 9). Moreover, there are also examples of flower life being prolonged by expressing a mutant ethylene receptor gene in chrysanthemums (see Non-Patent Document 10), flower form being changed by suppressing expression of chrysanthemum AGAMOUS gene (see Non-Patent Document 11), and expression of exogenous genes being increased in chrysanthemums by using a 5′-untranslated region of tobacco alcohol dehydrogenase (to also be referred to as tobacco ADH-5′UTR) (see Non-Patent Document 12).
On the other hand, although there have been examples of successful alteration of chrysanthemum flower color by genetic recombination, including a report of having changed pink flowers to white flowers by suppressing the chalcone synthase (CHS) gene by co-suppression (see Non-Patent Document 13), and a report of having changed white flowers to yellow flowers by suppressing carotenoid cleavage dioxygenase (CCD4a) by RNAi (see Non-Patent Document 14), all of these methods involve alteration of flower color by suppressing expression of endogenous genes, and there have been no successful examples of altering flower color by over-expression of exogenous genes as well as no examples of having realized a change in anthocyanin structure or an accompanying change in flower color.
Although attempts to alter flower color by over-expression of an exogenous gene have been reported that involve introducing a gene encoding F3′5′H, which is an enzyme required for synthesis of delphinidin (see Patent Document 5 and Non-Patent Document 15), the delphinidin produced due to the action of the introduced F3′5′H gene accumulates in ray petals, and there are no reports of the production of bluish chrysanthemums. In chrysanthemums, even if F3′5′H is expressed with CaMV35S promoter, production of delphinidin is not observed (see Non-Patent Document 15). In addition, expression of a gene expressed with CaMV35S promoter is unsuitable for stable expression, and for example, ends up dissipating accompanying growth of the chrysanthemum transformant (see Non-Patent Document 7). Potato Lhca3.St.1 promoter (see Non-Patent Document 16), chrysanthemum UEP1 promoter (see Non-Patent Document 17) and tobacco EF1α promoter (see Patent Document 6 and Non-Patent Document 9), for example, have been developed for use as promoters enabling efficient and stable expression of exogenous genes in the ray petals of chrysanthemums. However, there have been no reports describing alteration of chrysanthemum flower color by over-expression of an exogenous gene using these promoters. On the basis of the above, in order to produce chrysanthemums in which flower color has been altered by genetic recombination, it is necessary to establish a technology for controlling the expression of flavonoid biosynthesis genes, including the development of a promoter suitable for chrysanthemums. 